Method for enhancing accumulation of extracellular elemental sulfur by pseudomonas putida during degradation of propanethiol through gene knockout

Abstract

The invention discloses a method for strengthening accumulation of extracellular elemental sulfur by pseudomonas putida during degradation of propanethiol through gene knockout. The method comprises the following steps: by taking a propanethiol degrading bacterium Pseudomonas putida S-1 as an original strain, in a basic salt culture medium taking propanethiol as a unique carbon source, the S-1 can degrade propanethiol and generate separable extracellular elemental sulfur (sulfur balls) at the same time; extracellular sulfur ball accumulation can be enhanced by knocking out the persulfide dioxygenase coding gene in the S-1 strain, the yield of the extracellular elemental sulfur is increased by 69.2% compared with that of a wild type, and the yield of the elemental sulfur is increased while propanethiol is degraded by microorganisms.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for enhancing the accumulation of extracellular sulfur in *Pseudomonas putida* during the degradation of propanethiol by gene knockout. Background Technology

[0002] Propanethiol (PT) contains a thiol group (-SH) and is a typical volatile organic sulfur compound. It is characterized by high volatility, foul odor, and low olfactory threshold, threatening the ecological environment and human health.

[0003] Elemental sulfur has both industrial and agricultural value. It can be used as a raw material for vulcanized rubber and lithium batteries, and can also be used directly as agricultural fertilizer, making it widely applicable.

[0004] Propane mercaptan degradation includes physical, chemical, and biological methods. Biological methods are more efficient and eco-friendly compared to the former. Elemental sulfur can be produced through the catalytic reduction of sulfur dioxide using inorganic catalysts, but this process requires high temperatures and consumes significant energy. To meet the needs of volatile organic sulfur compound (VOC) remediation and resource utilization, constructing engineered strains using genetic engineering techniques to increase elemental sulfur production while simultaneously degrading propane mercaptan offers another sustainable environmental remediation approach. To date, there have been no reports of propane mercaptan degradation and simultaneous elemental sulfur production in *Pseudomonas putida*, nor have there been reports of promoting elemental sulfur accumulation during propane mercaptan degradation using gene knockout technology. Summary of the Invention

[0005] Based on the shortcomings of current methods, this invention proposes for the first time a method to enhance the accumulation of extracellular sulfur in *Pseudomonas putida* during the degradation of propanethiol by gene knockout.

[0006] The technical problem to be solved by this invention is to provide an engineered bacterial strain capable of simultaneously degrading propanethiol and generating elemental sulfur, while also addressing the technical issue of increasing the sulfur yield of this strain. This invention achieves the simultaneous degradation of propanethiol and promotion of increased sulfur yield through a gene knockout strategy, mainly through the following means: 1. Propanthiol-degrading bacteria *Pseudomonas putida* P. putida Starting with S-1, in a basic salt medium with propanethiol as the sole carbon source, S-1 can generate separable extracellular sulfur globules while degrading propanethiol. 2. Knocking out the gene encoding supersulfide dioxygenase in S-1 can promote the production of extracellular sulfur globules. The extracellular sulfur production of the knockout strain increased by 69.2% compared with the wild type, realizing the generation of sulfur while simultaneously degrading propanethiol by microorganisms and achieving increased production.

[0007] Specifically, the method for constructing the engineered strain of *Pseudomonas putida* includes the following steps: 1) Selection of strains and plasmids: *E. coli* DH5α was used for the construction and propagation of all plasmids; *E. coli* WM3064 was used for conjugation to construct the knockout strain; P. putida S-1 wild-type was used as the starting strain; 2) DNA manipulation: All upstream and downstream homologous arms of the knockout gene fragment were manipulated using... P. putida S-1 genomic DNA was used as a template for PCR amplification; sucrose-lethal plasmid pK18 was used. mobsacB As a knockout plasmid, the upstream and downstream homologous arms of the knockout gene were ligated to pK18 via Gibson assembly. mobsacB Gene knockout vectors are obtained from multiple cloning sequences.

[0008] 3) Strain construction: 3.1) A homologous recombination double-crossover gene knockout system was used, employing pK18 mobsacB The knockout plasmid was used to perform gene knockout genetic manipulation in the S-1 strain; the knockout vector was heat-shocked and transformed into Escherichia coli DH5α for plasmid amplification; the extracted plasmid was transformed into the 2,6-diaminopimelic acid-deficient strain Escherichia coli WM3064 with sex fimbriae; the correct transformant was combined with S-1, and the gene-deficient strain with seamless knockout was obtained through two homologous recombination screenings. 3.2) Increase sulfur production by knocking out the gene encoding persulfide dioxygenase; to avoid the impact of knockout on the promoters of adjacent genes, [the following measures are taken]. pdo0006 Perform partial knockout, i.e., knockout as pdo0006 The sequence from 517bp to 882bp was obtained; a gene knockout strain, namely S-1Δ, was constructed. pdo0006 .

[0009] The present invention also provides the engineered bacteria S-1Δ pdo0006 The application method in the simultaneous production of elemental sulfur from propanethiol degradation is as follows: The engineered strain S-1Δ... pdo0006 Inoculate into 20 mL LB medium and activate overnight in a shaker at 30°C; transfer the activated bacterial culture to 50 mL LB medium and culture in a shaker at 30°C until the logarithmic growth phase; use the initial bacterial concentration OD 600=0.6% was inoculated into 1 mL of basal salt medium (MM-PT) containing 100 mg / L propanethiol, and the propanethiol degradation reaction was carried out in a shake flask at 30℃ and 160 rpm. The composition of the basal salt medium was: anhydrous calcium chloride (CaCl2) 0.023 g / L, potassium dihydrogen phosphate (KH2PO4) 1.0 g / L, ammonium chloride (NH4Cl) 1.5 g / L, disodium hydrogen phosphate dodecahydrate (Na2HPO4∙12H2O) 4.5 g / L, magnesium chloride hexahydrate (MgCl2∙6H2O) 0.427 g / L, and 0.1% volume of trace element stock solution was added. The composition of the trace element stock solution was: sodium molybdate 0.02 g / L, ferrous chloride 1.00 g / L, boric acid 0.014 g / L, manganese chloride 0.01 g / L, zinc chloride 0.1 g / L, cobalt chloride hexahydrate 0.02 g / L. g / L, add ddH2O to bring the volume to 1 L.

[0010] The present invention also provides a product containing pdo0006 Application of wild-type *Pseudomonas putida* in the accumulation of extracellular sulfur during the degradation of propanethiol.

[0011] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following: the present invention utilizes microbial transformation to achieve the degradation of propanethiol while simultaneously producing elemental sulfur, and promotes the increase of elemental sulfur yield through genetic engineering, thus having good prospects for promotion and application. Attached Figure Description

[0012] Figure 1 Line graph showing the growth and degradation of *Pseudomonas putida* cultured with propanethiol as the sole carbon source; Figure 2 A bar chart showing the sulfur content of extracellular sulfur globules secreted by *Pseudomonas putida*. Detailed Implementation

[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0014] Obtaining relevant DNA fragments: Fragments approximately 500 bp upstream and downstream of the knockout gene were used as upstream and downstream homologous arms, and *Pseudomonas putida* was used as the target DNA fragment. P. putida Using the S-1 genome as a template, PCR amplification was performed using primers 0006UF, 0006UR, 0006DF, and 0006DR to obtain homologous arms as shown in SEQ ID NO.2 and SEQ ID NO.3. The upstream and downstream homologous arms of the knockout gene were then ligated to pK18 via Gibson assembly. mobsacB Gene knockout vectors are obtained from multiple cloning sequences.

[0015] The method used to construct the strain: Pseudomonas putida P. putidaS-1 generates elemental sulfur during the degradation of propanethiol, which is achieved through a homologous recombination double exchanger system. pdo0006 Gene knockout procedure. The knockout plasmid is transferred into the host cell via conjugation, and knockout is achieved without scarring through two homologous recombination screenings. PCR amplification is used to verify the success of gene knockout after each procedure.

[0016] Example 1

[0017] 1) A homologous recombination double-crossover gene knockout system was used, employing pK18. mobsacB The knockout plasmid was used for gene knockout genetic manipulation in strain S-1; the knockout vector was heat-shocked and transformed into *E. coli* DH5α for plasmid amplification; the extracted plasmid was transformed into *E. coli* WM3064, a 2,6-diaminopimelic acid-deficient strain with sex fimbriae; the correct transformants were then combined with *Pseudomonas putida*. P. putida S-1 conjugation, followed by two homologous recombination screenings, yielded gene-deficient strains with seamless knockout. 2) Increase sulfur production by knocking out the gene encoding persulfate dioxygenase; to avoid the impact of knockout on the promoters of adjacent genes, the nucleotide sequence is as shown in SEQ ID NO.1. pdo0006 Perform partial knockout, i.e., knockout as pdo0006 The sequence from 517bp to 882bp was used to obtain the gene with the nucleotide sequence described in SEQ ID NO.4; a gene knockout strain, namely S-1Δ, was constructed. pdo0006 .

[0018] The primer sequences used in the construction according to the embodiments of the present invention are shown in Table 1.

[0019] Table 1 Summary of Primers Used

[0020] Wild-type strain of Pseudomonas putida P. putida S-1 and engineered strain S-1Δ pdo0006 Inoculate each culture into 20 mL LB medium and activate overnight in a shaker at 30°C; transfer the activated culture to 50 mL LB medium and incubate with shaking at 30°C until the logarithmic growth phase; use the initial cell concentration OD 600=0.6% was inoculated into 1 mL of basal salt medium (MM-PT) containing 100 mg / L propanethiol, and the propanethiol degradation reaction was carried out in a shake flask at 30℃ and 160 rpm. The control group contained only the medium. The MM medium composition was: anhydrous calcium chloride (CaCl2) 0.023 g / L, potassium dihydrogen phosphate (KH2PO4) 1.0 g / L, ammonium chloride (NH4Cl) 1.5 g / L, disodium hydrogen phosphate dodecahydrate (Na2HPO4∙12H2O) 4.5 g / L, magnesium chloride hexahydrate (MgCl2∙6H2O) 0.427 g / L, with 0.1% volume of trace element stock solution added. The trace element stock solution composition was: sodium molybdate 0.02 g / L, ferrous chloride 1.00 g / L, boric acid 0.014 g / L, manganese chloride 0.01 g / L, zinc chloride 0.1 g / L, cobalt chloride hexahydrate 0.02 g / L. g / L, add ddH2O to make up to 1 L. The growth of the strain and the degradation of propanethiol can be observed. Figure 1 , Figure 1 In this context, A represents the growth curve. Figure 1 In the figure, B represents the degradation curve, which is derived from... Figure 1 It can be observed that there is basically no significant difference in growth and degradation between wild-type and knockout strains within 24 hours, and both can completely degrade 100 mg / L propanethiol within 24 hours.

[0021] Extracellular disulfocospheric compounds were separated from bacterial cells using a 2 M sucrose solution, followed by ultrasonic disruption, dichloromethane extraction and volatilization, and carbon disulfide extraction. The extract was then filtered and subjected to high-performance liquid chromatography (HPLC). The extract was analyzed on an Agilent 1260 HPLC instrument equipped with a reversed-phase C18 column (250 × 4.6 mm, 5 μm; Agilent, USA) and a UV detector, operated at 30 °C and a flow rate of 1.0 mL / min. The mobile phase was an aqueous solution of 95% methanol. The injection volume was 20 μL. Figure 2 As shown, the yield of elemental sulfur in the wild type was 8.17 μg / L, and the yield in the knockout strain was 13.82 μg / L.

Claims

1. A method for enhancing the accumulation of extracellular sulfur in *Pseudomonas putida* during the degradation of propanethiol by gene knockout, characterized in that, Includes the following steps: 1) Provide *Pseudomonas putida* P. putida S-1 wild-type was used as the starting strain; 2) Knock out the persulfide dioxygenase encoding gene in the starting strain from step 1). pdo0006 The sequence from 517bp to 882bp was used to construct the gene knockout strain S-1Δ. pdo0006 ; The gene knockout in step 2) uses a homologous recombination double crossover gene knockout system with plasmid pK18. mobsacB As a knockout vector, and assembled via Gibson, pdo0006 The upstream and downstream homologous arms of the gene are linked to the vector; pdo0006 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method for enhancing the accumulation of extracellular sulfur in *Pseudomonas putida* during the degradation of propanethiol by gene knockout as described in claim 1, characterized in that... The specific procedures for gene knockout in step 2) include: S1. with P. putida Using S-1 genomic DNA as a template, PCR amplification was performed. pdo0006 Upstream and downstream homologous arms of a gene; S2. The upstream and downstream homologous arms were ligated to plasmid pK18 via Gibson assembly. mobsacB Multiple cloning sites were identified to obtain gene knockout vectors; S3. The gene knockout vector was sequentially transformed into E. coli DH5α and WM3064, and then introduced via conjugative transfer. P. putida S-1 strain; S4. Through two rounds of homologous recombination screening, a gene-deficient strain S-1Δ with seamless knockout was obtained. pdo0006 .

3. An engineered strain S-1Δ constructed using the method described in claim 1 pdo0006 Its application in the degradation of propanethiol and the generation of elemental sulfur is characterized by, Includes the following steps: 1) Incorporate engineered strain S-1Δ pdo0006 Inoculate into LB medium and activate culture at 30°C; 2) Transfer the activated bacterial culture to fresh LB medium and culture with shaking until the logarithmic growth phase; 3) Using the initial bacterial cell concentration OD 600 =0.6% was inoculated into MM-PT basal salt medium containing propanethiol, and the propanethiol degradation reaction was carried out at 30℃ and 160 rpm. The basic salt culture medium consisted of: 0.023 g / L anhydrous calcium chloride, 1.0 g / L potassium dihydrogen phosphate, 1.5 g / L ammonium chloride, 4.5 g / L disodium hydrogen phosphate dodecahydrate, and 0.427 g / L magnesium chloride hexahydrate, with 0.1% volume of trace element stock solution added. The trace element stock solution consisted of: 0.02 g / L sodium molybdate, 1.00 g / L ferrous chloride, 0.014 g / L boric acid, 0.01 g / L manganese chloride, 0.1 g / L zinc chloride, and 0.02 g / L cobalt chloride hexahydrate, with ddH2O added to bring the volume to 1 L.

4. The application of a *Pseudomonas putida* strain in the accumulation of extracellular sulfur during the degradation of propanethiol, characterized in that... Pseudomonas putida P. putida S-1 contains the contents as described in claim 1 pdo0006 Gene.

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